Methods of forming bone interface scaffolds

ABSTRACT

Methods of forming a connective tissue-to-bone interface scaffolds (e.g., ligament-to-bone interface scaffolds, tendon-to-bone interface scaffolds, etc.). These scaffolds (grafts) may be formed from in such a way as to provide both a mineralized and demineralized layer in which the entire graft is flexible, compressible and compliant.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to U.S. Provisional PatentApplication No. 63/069,662, titled “METHODS OF FORMING BONE INTERFACESCAFFOLDS,” filed on Aug. 24, 2020, and herein incorporated by referencein its entirety.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

FIELD

Described herein are scaffolds for repair of the enthesis, includingligament to bone and tendon to bone interface scaffolds for repair oftorn tendons, ligaments, and/or calcified and uncalcifiedfibrocartilage.

BACKGROUND

Connective tissues, including tendons and ligaments, provide jointstability, guide joint motion, and play an important role inproprioception. Injury to ligaments and tendons represents a majorportion of all sports-related injuries on an annual basis. Inparticular, rotator cuff injuries are particularly prevalent anddifficult to repair.

The rotator cuff is a group of muscles and tendons that surround theshoulder joint, keeping the head of your upper arm bone firmly withinthe shallow socket of the shoulder. A rotator cuff injury can cause adull ache in the shoulder, which often worsens when you try to sleep onthe involved side. Rotator cuff injuries occur most often in people whorepeatedly perform overhead motions in their jobs or sports. Examplesinclude painters, carpenters, and people who play baseball or tennis,although rotator cuff tears may occur as a result of a single injury.The risk of rotator cuff injury increases with age.

Extensive rotator cuff tears typically require surgical repair, howevercurrent techniques are not optimal, and may be difficult or impossibleto achieve, or may fail days or months after surgery. In particular,reattachment of connective tissue, including rotator cuff tissue, tobone is notoriously difficult to achieve. In uninjured tissue, theinterface between the soft connective tissue and the bone occurs througha complex and distinct interface having multiple layers. The first isthe connective tissue proper, or midsubstance, which consists mostly ofa type I collagen matrix. The midsubstance inserts into a layer offibrocartilage mainly composed of type II collagen rich withproteoglycans. This layer transitions into calcified fibrocartilagelayer. The final region is subchondral bone, which contained amineralized type I collagen matrix. The junction between bone and softconnective tissue has controlled heterogeneity, permitting a gradualmanner of load transmission from the hard tissue to the soft tissue in amanner hypothesized to minimize stress and strain concentrations.Studies using autografts have shown that using the soft connectivetissue proper as the sole graft does not lead to strong biologicalintegration and the re-establishment of the native bone-soft tissueinterface. Without such integration, mechanical stability is limited atthe joint and the lack of integration can produce higher rates of graftfailure. In order to restore the physiological structure and function ofthe tissue, new strategies must be developed for the treatment of softconnective tissue ruptures.

Tissue engineering has emerged in the past twenty years as a promisingstrategy for soft connective tissue repairs. There have been a number ofreports on the use of tissue engineering techniques to regenerateligaments and tendons. However, most of these studies focus on themidsubstance region and fail to address the regeneration of theinterface. To date, collagen fibers, silk fibers, collagen gels andsynthetic polymer scaffolds have been utilized to replace the softtissue portion of the ligament or tendon. One example is a compositecollagen fiber-collagen gel scaffold seeded with fibroblasts that doesnot degrade in vitro and matches many of the mechanical properties ofnormal ligaments. Unfortunately for many tissues, especially those inthe musculoskeletal system, matching the mechanical properties is notsufficient. In order to transmit loads, the construct must successfullyintegrate with the host tissue and revascularize, processes that arelargely governed by the construct's permeability.

Current standard of care for reattaching connective tissues (ligament,tendon) to bone typically uses suture and suture anchors to reattach theconnective tissue to the bone. Re-tearing of the connective tissue iscommon, and occurs in over half of the cases. Further, full mobility andpain relief are not generally possible.

Although bone implants for repairing damaged ligaments and tendons frombone have been proposed (e.g., U.S. Pat. Nos. 6,776,800, 6,855,169,7,753,963, and 8,702,809), these implants have been difficult toimplement. In particular, these implants have required hinge regions orother techniques for making them flexible, often compromising theirstrength and structural integrity and requiring complex fabricationprocesses. Thus, there is a need for a connective tissue-to-boneinterface scaffolds that can easily be integrated into bone andrevascularize, as well as provide attachment and ingrowth for theconnective tissue. In particular, it is desirable to provide scaffoldsthat may be adapted for easy attachment and use.

SUMMARY OF THE DISCLOSURE

Described herein are connective tissue-to-bone interface scaffolds(e.g., ligament-to-bone interface scaffolds, tendon-to-bone interfacescaffolds, etc.). These scaffolds may be referred to as grafts orimplants, and may be a single integrated implant or may be a modular(e.g., two- or more part) implant system.

The connective tissue-to-bone interface grafts described herein aretypically formed of a bone material that has been processed to form alayered structure having a first layer that is demineralized and porousand a second layer that is mineralized and porous. The graft may be astrip, having an elongate, flattened sheet or strip-like configuration,in which the first (e.g., upper) major side is mineralized and thesecond (e.g., lower) major side is demineralized. The edges between thefirst and second side (minor sides) may be rounded or beveled.

For example, in some examples the connective tissue-to-bone interfacegraft described herein may be formed of a bone allograft material thatis derived from human bone (e.g., cancellous bone) and may have a lengthof between, e.g., 10-50 mm, a width of between 6-26 mm in width, and athickness of between 1-6.5 mm. In some examples the physician may cutthis shape (trimming the length and/or width) down to a different lengthand width for implantation. As mentioned, the second major side isdemineralized to remove calcium and may be treated to preventosteogenesis, allowing the connective tissue (e.g., in some examples ofthe rotator cuff) to heal directly to the implant on the demineralizedside. The demineralized side may be formed mostly of type I collagen,similar to the tendon material. The opposite, first major side mayremain mineralized, providing a hard, tissue-facing layer that enablesosseointegration and fixation to the bone. The top side and the bottomside are typically continuous with each other and may be formed of thesame original strip of material (e.g., formed of a unitary section ofcancellous bone). Alternatively, in some examples the first layer(demineralized) and the second (mineralized) layers may be separatelyformed and attached, e.g., by an adhesive having the biocompatibility,strength and viscosity necessary to allow it to hold the mineralizedportion to the demineralized portion without occluding the pores oneither side.

As described herein, it may be particularly important to preventoccluding of the pores in either or both the mineralized anddemineralized sides of the implant. The grafts described hereintypically have a high porosity, allowing fluid conductance and rapidcell incorporation into the thickness of the graft. The porosity alsopromotes vascular ingrowth.

In use, the graft is affixed to the bone. For example, the bone site(the site of implantation) may be prepared first to receive the implantby exposing the bone and marrow in an attachment region (e.g., a cut-outregion), then one or more anchors may be inserted into the humerus atthe medial edge of the rotator cuff, then the scaffold may be attachedto the anchors with the bone ingrowth side (the mineralized side) facingthe bone. The graft may be secured in place further by suture knots(tying knots over the scaffold).

In general, it may be particularly beneficial to have the graft beconfigured to flexibility and compliance, so that it may be implanted,e.g., through a cannula, by folding or curling it, and also for allowingthe graft to conform to the often irregular surface of the cut-ourregion of the bone. As described herein, the applicants havesurprisingly found that this may be achieved by controlling thethickness of the mineralized region, e.g., so that it is much thinnerthan the demineralized region, and not greater than about 1.5 mm (e.g.,is 1.5 mm or less, 1.4 mm or less, 1.3 mm or less, 1.2 mm or less, 1.1mm or less, 1.0 mm or less, 0.9 mm or less, 0.8 mm or less, etc.). Thus,in general, the thickness of the mineralized layer may be different andoften much less than the thicknesses of the demineralized layer formingthe rest of the graft. For example, the thickness of the demineralizedportion of the implant may be 55-99% (e.g., between about 60% and 99%,between about 60-95%, between about 55-95%, between about 55-80%,between about 55-75%, etc.) of the thickness of the graft (e.g., theimplant or scaffold).

Although in some of the grafts described herein the body of the graftmay be sufficiently ‘soft’ so that a needle may be passed through it,e.g., puncturing it, in any of the grafts described herein may beconfigured for insertion or implantation into the bone. For example, anyof these implants may include one or more pre-formed suture holes orpassages through or into the body of the implant. These suture openingsor holes (or in some examples, channels) may be configured to allowsuture to be threaded into and through the implant so that they bereadily secured into the body, particularly into the bone tissue. Insome examples the implant may be pre-threaded with one or more lengthsof suture, which may greatly enhance the ease of use and may help guidethe physician in attaching and positioning the implants. The pre-formed(e.g., drilled) holes may be treated to allow the suture to pass throughbut may prevent tearing or damage to the graft, particularly the softer,demineralized side.

Also described herein are multi-part grafts that may including multipleplanar layers that are either preassembled (e.g., using an adhesive,suture, etc.) or may be used separately and attached. For example, theimplants described herein may include a first layer that is mineralizedthat is attached (e.g., via an adhesive) either directly to a secondlayer of demineralized bone that may be complimentary to the firstlayer, or indirectly, e.g., through a third, intermediate layer. In someexamples the first and second (or first, second and third) layers may beconfigured to engage with each other, e.g., via a formed attachmentbetween the complimentary sides of the first and second (or first andthird, second and third) layers. For example, the first layer mayattachably couple to the second layer.

In general the grafts described herein may have a low stiffness (e.g.,may be highly flexible), and a high compressibility and compliance. Theflexibility and compliance may allow the graft to be inserted minimallyinvasively through a cannula or other introducer, despite having amineralized outer surface that can interface with the bone. The highcompliance of the graft may also allow it to conform to the bone (e.g.,an attachment/cut-out region of the bone) more readily, which mayimprove outcomes.

For example, described herein are flexible, compressible and compliantgrafts that may comprise a strip of bone forming a body having a length,a width and a thickness, wherein the thickness is less than half thelength and half the width, the body further comprising: a first layer ofdemineralized bone extending the length and the width of the body; and asecond layer of mineralized bone that is continuously adjacent to thefirst layer, wherein the second layer has a thickness that is less than1.5 mm. In some examples the second layer is between about 1.2 mm and0.1 mm thick. The second layer typically forms an outer surface of thegraft. The second layer may have a substantially constant thickness,e.g., the thickness may vary by less than 10% (e.g., less than 9%, lessthan 8%, less than 7%, less than 6%, less than 5%, less than 4%, lessthan 3%, etc.) along the entire second layer. The thickness of the bodymay be, for example, between about 3.5 and about 6.5 mm. In any of thesegrafts, the density of the graft (e.g. the density of the body) may beless than 2.6e-4 g/mm³.

For example, a flexible, compressible and compliant graft, may comprisea strip of bone forming a body having a length, a width and a thickness,wherein the thickness is between 4 mm and 6.5 mm and is less than halfthe length and half the width, the body further comprising: a firstlayer of porous demineralized bone extending the length and width of thebody; and a second layer of porous mineralized bone extending along anouter surface of the strip that is continuously adjacent to the firstlayer, wherein the second layer has a constant thickness that is lessthan 1.5 mm; wherein the body has a density that is less than 2.6e-4g/mm³.

For example, a flexible, compressible and compliant graft may comprise astrip of bone forming a body having a length, a width and a thickness,wherein the thickness is between 4 mm and 6.5 mm and is less than halfthe length and half the width, the body further comprising: a firstlayer of porous demineralized bone extending the length and width of thebody; and a second layer of porous mineralized bone extending along anouter surface of the strip that is continuously adjacent to the firstlayer, wherein the second layer has a thickness that is less than 1.5 mmthat varies by less than 10% along the entire second layer; wherein thebody had a density that is less than 2.6e-4 g/mm³ and a mean bendingthickness that is less than about 1.2 mNm.

The body of the graft may have a mean bending thickness that is lessthan about 1.2 mNm (e.g., less than about 1.1 mNm, less than about 1.0mNm, less than about 0.9 mNm, less than about 0.8 mNm, less than about0.7 mNm, less than about 0.6 mNm, less than about 0.5 mNm, less thanabout 0.1 mNm, etc.). Thus, the graft, particularly when hydrated priorto implantation, may be highly flexible, comparable to paper ornewsprint, without breaking or disrupting the mineralized layerextending across the side.

As mentioned, any of these grafts may include one or more suturechannels pre-formed through the thickness of the body.

The bone forming the graft may be any appropriate bone, including human(allograft) or non-human bone, such as porcine cancellous bone.

The edges of the body of the graft may be square or may be rounded,e.g., having a radius of curvature of between 0.25 mm and 2 mm. In someexamples the edges of the body are beveled at an angle of between 30 and60 degrees.

In any of the grafts described herein the first layer may have adifferent color than the second layer. This may allow immediate and easyrecognition of the mineralized vs.

demineralized sides of the graft, which may otherwise be difficult todistinguish. In some examples a biocompatible dye or coloring is used tomark or label one or both sides to indicate which is which.

As mentioned, the graft body may have a length of, e.g., between about15 and 50 mm, and a width of, e.g., between about 10-25 mm. In general,the graft body may be substantially free of fat and blood proteins. Insome examples the first layer (e.g., the demineralized layer) issubstantially free of ostioinductivity.

In some examples, the mineralized (e.g., second) layer has 8% or morecalcium, while the demineralized (first) layer has less than 8% calcium.

As mentioned, also described herein are methods of using any of thesegrafts to repair tissue, including (but not limited to) repair of arotator cuff. For example, a method of repairing a rotator cuff mayinclude: removing a region of a cortical layer of a humerus and/orforming microfractures in the humerous to expose a marrow material in anattachment region; anchoring one or more suture to the humerus; passingthe one or more sutures through a thickness of a flexible, compressibleand compliant graft, the graft having a length, a width and a thickness,wherein the thickness is less than half the length and half the width,the graft further comprising: a first layer of demineralized bone and asecond layer of mineralized bone that is continuously adjacent to thefirst layer, wherein the second layer has a thickness that is less than1.5 mm; securing the second layer of the graft in contact with themarrow material, wherein the second layer compliantly conforms to theattachment region (in some examples, so that fluid pressure from thehumerous drives the marrow material to infiltrate the second layer); andsuturing a tendon against the first layer. The thickness of the secondlayer may be between 1.2 mm and 0.1 mm. The graft may have a densitythat is less than 2.6e-4 g/mm³.

The step of removing a region of the cortical layer and/or formingmicrofractures in the humerous may be done alternatively oradditionally. For example, in some examples, the bone (e.g., humerous)is prepared by removing a region of the cortical layer, e.g., to form ashelf or landing pad region to which the graft may be attached.Alternatively, in some examples the bone (e.g., humerous) may beprepared by forming microfractures to the region to which the graft willbe attached, without removing a region. In some examples both a regionof the bone may be removed and the bone may be treated to formmicrofractures.

For example, a method of repairing a rotator cuff may include: removinga region of a cortical layer of a humerus and/or forming microfracturesin the bone to expose a marrow material within an attachment region,wherein the region has a length of between 10 and 50 mm, a width ofbetween 6 and 25 mm; anchoring one or more suture to the humerus;placing a flexible, compressible and compliant graft against theattachment region, the graft having a length, a width and a thickness,wherein the thickness is less than half the length and half the width,the graft further comprising: a first layer of demineralized bone and asecond layer of mineralized bone that is continuously adjacent to thefirst layer, wherein the second layer has a thickness that is less than1.5 mm; securing the graft to the humerus so that the second layer ofthe graft in contact with the marrow material and compliantly conformsto the attachment region, so that fluid pressure from the humerousdrives the marrow material to infiltrate the second layer; and suturinga tendon against the second bone scaffold.

Suturing the tendon against the first layer may comprise suturing withthe one or more sutures. Suturing the tendon may comprise suturing thetendons of one or more of: the supraspinatus muscle, infraspinatusmuscle teres minor muscle or the subscapularis muscle.

Passing the one or more sutures through the thickness of the graft maycomprise passing the one or more suture through holes pre-formed throughthe graft. Alternatively or additionally, the grafts described hereinmay permit a needle to be passed through the graft without requiring apre-formed hole.

In general, described herein are method, and in particular, methods offorming a compressible and compliant graft from a porous bone. Forexample, a method of forming a compressible and compliant graft asdescribed herein may include: cutting donor bone tissue into a thinlayer having a thickness; applying a masking agent to a first side of abody of the donor bone tissue to a thickness of less than 1.5 mm,forming a masked portion and an unasked portion, wherein the maskingagent comprises a cyanoacrylate having a polar side chain including anoxygen; demineralizing the unmasked portion of the donor bone tissue;and removing the masking agent to form a graft comprising a first layerof demineralized bone extending a length and a width of the body; and asecond layer of mineralized bone that is continuously adjacent to thefirst layer, wherein the second layer has a thickness that is less than1.5 mm.

The masking agent may be particularly important, particularly whenforming the relatively thin grafts described herein, as known maskingagents may either wick up and/or into the bone in a manner that mayovermask the bone, or may fail to reliably impregnate and mask the bone(undermasking the bone), or may be too difficult to remove, or maydamage the bone and/or the microstructure of the bone graft materialduring processing.

The inventors have identified a particular class of masking agents thatwork where other, similar compositions of masking agents, do not work.In particular, the methods descried herein may include a masking agentcomprising cyanoacrylate having a polar side chain including an oxygen.In particular, the masking agent may comprise ethoxyethyl cyanoacrylate.The cyanoacrylate having a polar side chain including an oxygen may beselected from the group consisting of ethoxyethyl cyanoacrylates,methoxypropyl cyanoacrylates and ethoxymethyl cyanoacrylates. Thecyanoacrylate having a polar side chain including an oxygen may beselected from the group of cyanoacrylates having a low blooming andrelatively quick curing properties.

Any of the masking agents described herein may include a thickener toreduce viscosity. The thickener may comprise polymethyl methacrylate(PMMA). The concentration of PMMA in the masking agent may be betweenabout 4% and about 12%. The PMMA may include an average molecular weightof about 350,000 or less.

In general, cutting the donor bone tissue into a thin layer having athickness may comprise cutting to a thickness of less than 5.5 mm.

Any of these methods may include washing the thin layer of donor bonetissue to remove organic material by exposing the cut donor bone tissueto a detergent including a protease. Alternatively or additionally, anyof these methods may include washing the thin layer of donor bone tissueto remove organic material by exposing the cut donor bone tissue to adefatting agent. For example, the defatting agent may comprise acetone.

Any of these methods may include trimming the cut donor bone tissue to alength of between about 15 and 50 mm, and a width of between about 10-25mm.

Applying the masking agent may comprise dipping the cut donor bonetissue into the masking agent for less than 1 minute to a depth of lessthan 1.5 mm. Any of these methods may include drying the masking agentfor 1 hour or less. Removing the masking agent may include rinsing thecut donor bone tissue in acetone.

Any of these methods may include dehydrating the graft for storage,and/or storing the graft after dehydration. The graft may be stored in asealed package (e.g., a foil package). To use the graft, it may berehydrated (e.g., in water).

Any appropriate donor bone tissue may be used, including in particularcancellous human bone.

The methods described herein may also include confirming a density orporosity of the cut donor bone tissue before applying the masking agent.

For example, a method of forming a compressible and compliant graft mayinclude: cutting donor bone tissue into a thin layer having a thicknessof less than 5.5 mm; removing organic material from the cut donor bonetissue; applying a masking agent to a first side of the donor bonetissue to a thickness of less than 1.5 mm, forming a masked portion andan unmasked portion, wherein the masking agent comprises an ethoxyethylcyanoacrylate; demineralizing the unmasked portion of the donor bonetissue; and removing the masking agent to form a graft comprising a bodyhaving a length, a width and a thickness that is less than half thelength and half the width, the body further comprising: a first layer ofdemineralized bone extending the length and the width of the body; and asecond layer of mineralized bone that is continuously adjacent to thefirst layer, wherein the second layer has a thickness that is less than1.5 mm.

Also described herein are methods of forming a compressible andcompliant graft from a porous bone, the method comprising: applying amasking agent to a first side of a body comprising a donor bone tissueto a thickness of less than 1.5 mm, wherein the masking agent comprisesa cyanoacrylate having a polar side chain including an oxygen;demineralizing an unmasked second side of the donor bone tissue, whereinthe second side is opposite and adjacent the first side; and removingthe masking agent to form a graft comprising a first layer ofdemineralized bone extending a length and a width of the body; and asecond layer of mineralized bone that is continuously adjacent to thefirst layer, wherein the second layer has a thickness that is less than1.5 mm.

All of the methods and apparatuses described herein, in any combination,are herein contemplated and can be used to achieve the benefits asdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe claims that follow. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is an example of a bone scaffold having a layer of demineralizedand a layer of mineralized bone as described herein.

FIGS. 2A-2E illustrate example of biocompatible bone scaffolds asdescribed herein.

FIG. 3 is an example of a bone scaffold including pre-formed suturechannels.

FIGS. 4A-4B illustrate bone scaffolds having rounded (FIG. 4A) ortapered (FIG. 4B) edges as described herein.

FIGS. 5A-5B show examples of bone scaffolds having initially separatedemineralized and mineralized layers.

FIGS. 6 (1)-6(10) illustrates one method of implanting/inserting bonescaffold to repair a connective (e.g., tendon) tissue as describedherein.

FIGS. 7A and 7B illustrate examples of the decalcified (FIG. 7A) andcalcified (FIG. 7B) sides of a graft as described herein.

FIG. 7C shows a side view of a graft as described herein, including bothcalcified and decalcified layers, positioned adjacent to each other.

FIG. 7D shows an example of a graft (such as the one shown in FIG. 7C)being bent between two fingers.

FIGS. 7E and 7F illustrate the compressibility of the graft shown inFIG. 7D. FIG. 7E shows the graft between two fingers in an uncompressedconfiguration. FIG. 7F shows the same graft being compressed between twofingers.

FIG. 8 illustrates one method of forming a bone scaffold graft asdescribed herein.

DETAILED DESCRIPTION

Described herein are connective tissue-to-bone interface scaffolds(e.g., grafts) that are adapted and configured for surgical implantationonto bone to allow ingrowth of both bone as well as connective tissue.These grafts may be particularly well adapted for implantation andattachment as part of a minimally-invasive, e.g., arthroscopic,laparoscopic, surgery.

For example, FIG. 1 shows one example of a tissue-to-bone interfacescaffold as described herein 100. In FIG. 1 , the scaffold includes afirst, demineralized layer 101 and a second mineralized layer 103. Aswill be described in detail below, the dimensions of the mineralized anddemineralized layers, and in particular, their ratios, may be selectedwithin a desired range to optimize both the ease of using them in theparticular surgical procedures described herein, as well as forstability and in-growth of connective tissue and/or bone. In FIG. 1 themineralized and demineralized portions are both porous, although theymay have different porosities. For example, the porosity of themineralized side of the implant may be greater than (or the same as) theporosity of the demineralized side, and may be configured or selected topermit the fluid pressure from the humerus to drive bone marrow into thescaffold (e.g., into the cancellous side of the scaffold). In someexamples the implant may be configured to limit the migration of thebone marrow into the demineralized side, e.g., by limiting the porosityand/or by treatments to limit migration (including treatments to reducethe side, modify the surface properties, provide a barrier to migration,etc.). In some examples the scaffold may be configured to permitmigration of the bone marrow into the demineralized side.

In general the bone scaffold may be at least two layers, e.g., thedemineralized layer and the mineralized layer, that are immediatelyadjacent to each other. The scaffold may be shaped and sized so that itmay be inserted or implanted into the body, as will be described ingreater detail below, as well as providing optimal attachment to thebone and connective tissue.

For example, as shown in FIGS. 1-2D, the biocompatible bone scaffold maybe rectangular or approximately rectangular (e.g., rectanguloid), andmay have one or more rounded edges. In some examples the scaffold has astructure dimensioned somewhat like a stick of gum, e.g., having a flatbody that is longer than it is wide, and substantially thinner than itis wide or long. For example, the length and/or width may be greaterthan 2× (e.g., greater than about 3×, 4×, 5×, 10×, 15×, 20×, etc.) thethickness of the scaffold, where the thickness is the combined thicknessof the mineralized and demineralized layers. The mineralized anddemineralized layers may be the same length and width; in some examplesthe mineralized and demineralized layers have different lengths and/orwidths but either the width and/or the length of the demineralized layermaybe within +/−20% (e.g., +/−18%, +/−15%, +/−12.5%, +/−10%, +/−8%,+/−7.5%, +/−5%, etc.) of the mineralized layer.

Thus, the scaffolds described herein typically comprise a collagen-basedporous network capable of guiding tissue differentiation that can beused to regrow damaged soft tissues (e.g., connective tissue). Therelatively high porosity, e.g. of either or both the demineralized andmineralized material may allow host integration, regeneration ofrelatively large sections of tissue, and vascularization. Thecollagen-based porous structure may allow binding of a variety offactors to the trabeculae (pores) within the scaffold formed of bone.Additional materials, such as hydrogels or extracellular matrixmaterial, and/or a variety of biological components and therapeuticcompounds may be integrated within the scaffold. The scaffolds maycontain collagen trabeculae that may allow the structure to maintain apre-defined shape and maintain nutrient transport. Thus, the scaffoldsdescribed herein may desirably provide mechanical integrity, nutrienttransport during tissue regeneration, differentiation of well-definedcell populations, vascularization.

The grafts described herein may therefore include demineralized bone.The demineralized bone may be cancellous or corticocancellous bone.Cortical bone is the dense surface layer of the bone having littlevascularization. In contrast, cancellous bone is a spongy material thatmakes up the bulk of the interior of bones. Compared to cortical bone ithas a low density and strength, but very high surface area. Thesedifferences result in demineralized bone having differing properties,with demineralized cancellous bone comprising pores with diameters ofabout 100 microns to 2 mm while, in contrast, demineralized corticalbone may have a maximum pore size on the order of about 10 nm to 50microns.

The term “biocompatible” may refer to any material having a relativelylow risk of provoking an adverse response when introduced in a mammal,in particular a human patient. For example, a suitable biocompatiblematerial when introduced into a human patient has relatively lowimmunogenicity and toxicity. The term “demineralized” may refer to bonefrom which a substantial portion of minerals natively associated withthe bone minerals have been removed. The term “demineralized bone” isintended to refer to any bone, including cortical and/or cancellousbone, from any source including autologous, allogeneic and/or xenogeneicbone, that has been demineralized to contain, in certain examples, lessthan about 8 wt % residual calcium (e.g., less than about 7 wt %residual calcium, less than about 6 wt % residual calcium, less thanabout 5 wt % residual calcium, less than about 4 wt % residual calcium,less than about 3 wt % residual calcium, less than about 2 wt % residualcalcium, or less than about 1 wt % residual calcium, etc.).

A scaffold is “substantially free of mineralized bone” when all of thebone within the scaffold has been exposed to demineralizing conditionsand is at least partially demineralized. Scaffolds that aresubstantially free of mineralized bone are structurally and functionallydistinct from bioscaffolds made from bone that has been masked prior todemineralization (see, e.g., U.S. Published Application No.2011/0066241).

The term “osteoconductive” may refer to the ability of a substance tosupport or conduct bone growth, while “osteoinductive” may refer to theability of a substance to induce bone growth.

In some examples all or a portion of a scaffold as described herein maybe comprised of demineralized cancellous bone and/or portions, regions,or segments of demineralized cancellous bone that have been stiffened byphysicochemical methods, such as heating or stretching (i.e., strainhardening), or by crosslinking (e.g., chemically and/or physically) toincrease their strength, e.g., to hold sutures, to aid in retention ofshape, and/or to resist compression.

The bone scaffolds described herein may in certain examples be orinclude an autograft, an allograft or a xenograft. If the scaffold is axenograft it may be from, by way of non-limiting example, ovine, porcineor bovine bone. The bone may be taken from any bone having suitableproperties for the intended application of the scaffold. Properties toconsider in selecting bone for the bioscaffold include porosity, poresize, connectivity, mechanical strength, surface area/volume ratio, thesize of the scaffold required in the application, and the like. In someexamples, as will be described below, the bone may include both regionsof cancellous and cortical bone; thus the scaffolds may be derived fromcortical and/or cancellous bone. In certain examples, the bone isvertebral, femoral, or pelvic cancellous bone. In certain examples, thescaffold may be made from a continuous piece of bone. In certainexamples, the scaffold may be formed from multiple pieces of bone joinedtogether, for example, by suturing, crosslinking, adhesively connecting,etc.

A bone material for use as a scaffold may be treated to remove cells(including marrow). The section of bone may then be shaped using methodsknown in the art. Alternatively, the bone may be shaped before removalof the marrow. The bone section may be shaped into any shape desired forthe scaffold, particularly those described herein.

Bone may be demineralized in any appropriate manner. For example,demineralized bone may be formed by one or more of: decalcification byacid extraction; sonication in detergent solution (e.g., TERGAZYME®,Alconox, White Plains, N.Y.); alternated with rinsing in pure water (aswould be understood by the skilled artisan, this cycle may be repeatedas needed until substantially all fat, marrow, and other components inthe trabecular space are removed); treatment with alkylammonium salts ofEDTA, defatting by soaking in acetone; treatment with hydrochloric acid(HCl), in certain examples with ethylene diamine tetraacetic acid(EDTA). In certain examples, the demineralization process may includetreatment with one or more nonionic detergents, such as TRITON® X-100,Tween® 80, N,N-Dimethyldodecylamino-N-oxide, Octylglucoside,Polyoxyethylene (PEG) alcohols, Polyoxyethylene-p-t-octylphenol,Polyoxyethylene nonylphenol, Polyoxyethylene sorbitol esters,Polyoxy-propylene-polyoxyethylene esters, andp-isoOctylpolyoxy-ethylene-phenol formaldehyde polymer.

In certain examples, the bone scaffolds may be washed in peroxide (e.g.,H₂O₂) to remove osteoinductive factors. Other methods and reagents forremoving osteoinductive factors are known in the art, and include thosedescribed in U.S. Pub. No. 2005/0136124. Osteoinductivity of resultingscaffolds can be determined using standard methods in the art, such asELISA for BMP or other factors that contribute to osteoinductiveactivity (e.g., fibroblast growth factor-2 (FGF-2), insulin-like growthfactor-I and -II (IGF-I and IGF-II), platelet derived growth factor(PDGF), and transforming growth factor-beta 1 (TGF-β1)), on eluatesduring/after the treatment process.

One or more agents may be added to all or a portion of the scaffoldsdescribed herein. For example in some examples the demineralized ormineralized layers (or both) may include an agent to enhance ingrowthand/or attachment to the connective tissue and/or the bone. In someexamples, the bone scaffold (either or both the demineralized ormineralized layers) may be embedded with, injected with or otherwisehave attached thereto, cells, any of a variety of pharmaceuticals,antibiotics, growth factors, hydrogel, collagen gel or mixtures thereof.It is contemplated that any composition, compound or biologic that helpsin healing and integration of the scaffold may be added. Non-limitingexamples of cells that may be added to a scaffold include any variety ofstem cell, such as adult stem cells or cells derived from the softtissue to be repaired (e.g., cells from soft tissue including tendon,ligament, fascia, fibrous tissues, fat, synovial membranes, cartilagetissue, meniscal tissue, ligament tissue, tendon tissue, andcombinations thereof) or mixtures thereof. The tissue used can beautogeneic tissue, allogeneic tissue, or xenogeneic tissue. Tissueand/or cells can be obtained using any of a variety of conventionaltechniques, for example, by biopsy or other surgical removal.Preferably, the tissue sample is obtained under aseptic conditions.

Any of these scaffolds may have embedded therein, are embedded in,injected with, encapsulated by or otherwise attached to one or morepolymeric carriers and/or matrices which may be adapted to contain andrelease a compound or cell type of interest. For example, in someexamples the scaffold, and particularly the demineralized side, may beseeded with tendon progenitor cells. In certain examples, the carriercontaining the compound is a combination with a carbohydrate, protein orpolypeptide. Within certain examples, the polymeric carrier contains orcomprises regions, pockets, or granules of one or more of the compounds.For example, within one example, compounds may be incorporated within amatrix which contains the compound, followed by incorporation of thematrix within the polymeric carrier. A variety of matrices can beutilized in this regard, including for example, carbohydrates andpolysaccharides such as starch, cellulose, dextran, methylcellulose, andhyaluronic acid, proteins or polypeptides such as albumin, collagen andgelatin.

In some examples, the scaffolds described herein include a biocompatiblelayer. Such biocompatible layers may be semipermeable or bioresorbable.In other examples, scaffolds may be embedded in or encapsulated by abiodegradable layer. Such biocompatible and/or biodegradable layersinclude biodegradable polymers. For example, in certain examples,poly(c-caprolactone) (PCL) may be used with the scaffolds describedherein. PCL is an aliphatic polyester which can be degraded byhydrolysis under physiological conditions and it is non-toxic and tissuecompatible. The degradation of PCL is significantly slower than that ofcertain polymers and copolymers of lactic and glycolic acids and istherefore suitable for the design of long-term drug delivery systems.Other illustrative biodegradable polymers include, chitosan, heparin,chitosan-heparin complexes, biodegradable polymers, such as poly(DL-lactide-coglycolide) for sustained release delivery afterimplantation (Emerich, D F et al., 1999, Cell Transplant, 8, 47-58) orcompositions comprising polybutylcyanoacrylate. In certain examples,bioresorbable polycaprolactone/polyglycolic acid (PCL/PGA) polymers aresuitable. Examples of other biodegradable polymers include polymers orcopolymers formed from monomers of lactide, glycolide, dioxanone, andcaprolactone; collagen, fibrin, and silk; poly-(orthoesters) andpoly-(anhydrides), polylactic acid, polyglycolic acid, copolymers ofpolylactic and polyglycolic acid (e.g., poly(lactic-co-glycolic acid;PLGA), polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates andcross linked or amphipathic block copolymers of hydrogels. As usedherein, the term “glycolide” is understood to include polyglycolic acid.Further, the term “lactide” is understood to include L-lactide,D-lactide, blends thereof, and lactic acid polymers and copolymers.Biocompatible layers may be applied to the scaffold, by a variety ofwell-known techniques. For illustration, heparin can be applied to thescaffold in various ways including: First, benzalkonium heparin (BA-Hep)solution can be applied to the scaffold by dipping the scaffold in thesolution and then air-drying it. This procedure treats the scaffold withan ionically bound BA-Hep complex. Second, EDC can be used to activatethe heparin, then to covalently bond the heparin to the scaffold. Third,EDC can be used to activate the collagen, then covalently bond protamineto the collagen and then ionically bond heparin to the protamine. Manyother coating, bonding, and attachment procedures are well known in theart and may also be used. Treatment of the scaffold with drugs inaddition to or in substitution for heparin may be accomplished asdescribed elsewhere herein and based on art-established techniques.

In some examples the scaffolds described herein may also oralternatively include radio opaque materials (e.g., barium sulfate) thatwill render the scaffolds radio opaque. The scaffolds may also includematerials that will promote tissue regeneration or regrowth, as well asthose that act as buffers, reinforcing materials or porosity modifiers.As mentioned, the bone scaffolds described herein may be embedded withor otherwise comprise any of a variety of biomolecules, growth factors,differentiation factors, and like biological components. Any agent thatfacilitates tissue repair is contemplated for use with the scaffoldsdescribed herein. The biological components used in the scaffolds canalso be selected from among a variety of effectors that, when present atthe site of injury, promote healing and/or regeneration of the affectedtissue. In addition to being compounds or agents that actually promoteor expedite healing, the effectors may also include compounds or agentsthat prevent infection (e.g., antimicrobial agents and antibiotics),compounds or agents that reduce inflammation (e.g., anti-inflammatoryagents), compounds that prevent or minimize adhesion formation, such asoxidized regenerated cellulose (e.g., INTERCEED® and SURGICEL®,available from Ethicon, Inc.), hyaluronic acid, and compounds or agentsthat suppress the immune system (e.g., immunosuppressants). Thecomponent or effector compounds included with the scaffolds describedherein can be incorporated within the scaffold before or aftermanufacture of the scaffold, or before or after the surgical placementof the scaffold. Prior to surgical placement, the biocompatible scaffoldcan be placed in a suitable container comprising the biologicalcomponent. After an appropriate time and under suitable conditions, thescaffold will become impregnated with the biological component.Alternatively, the biological component can be incorporated within thescaffold by, for example, using an appropriately gauged syringe toinject the biological agent(s) into the scaffold. Other methods wellknown to those of ordinary skill in the art can be applied in order toload a scaffold with an appropriate biological component, such asmixing, pressing, spreading, centrifuging and placing the biologicalcomponent into the scaffold. Alternatively, the biological component canbe mixed with a gel-like carrier prior to injection into the scaffold.The gel-like carrier can be a biological or synthetic hydrogel asdescribed elsewhere herein, and/or may include an alginate, acrosslinked alginate, hyaluronic acid, collagen gel,poly(N-isopropylacrylamide), poly(oxyalkylene), a copolymer ofpoly(ethylene oxide)-poly(propylene oxide), poly(vinyl alcohol) andblends thereof. In some examples, following surgical placement, animplant wherein the biocompatible scaffold is devoid of any biologicalcomponent can be infused with biological agent(s), or an implant whereinthe scaffold includes at least one biological component can be augmentedwith a supplemental quantity of the biological component. One method ofincorporating a biological component within a surgically installedimplant is by injection using an appropriately gauged syringe.

Further agents for use with the scaffolds described herein include anyone or more of a variety of antibiotics. Antibiotics are well known inthe art and include Abacavir, Acyclovir, Albendazole, Amikacin,Amoxicillin, Ampicillin, Azithromycin, Aztreonam, Benzilpenicillin,Cefepime, Ceftriaxone, Cephalexin, Chloramphenicol, Chloroquine,Cilastatin, Clindamycin, Co-trimoxazole, Didanosine, Dioxidine,Doxycycline, Famciclovir, fluoroquinolones, Fluconazole, Fosfomycin,Furazolidone, Fusidic acid, Ganciclovir, Gentamicin, Isoniazid,Josamycin, Kanamycin, Ketoconazole, Lamivudine, Lincomycin, Linezolid,Mebendazole, Meropenem, Metronidazole, Moxifloxacin, Mupirocin,Nystatin, Nitrofurantoin, Nitroxoline, Norfloxacin, Ofloxacin,Ornidazole, Oseltamivir, Polymixin B, Polymyxin M, Proguanil, Ribavirin,Rifampicin, Rimantadine, Roxithromycin, Spectinomycin, Sulfodimidin,Teicoplanin, Terbinafine, Tetracycline, Timidazole, Valaciclovir,Valganciclovir, Vancomycin, Zanamivir, and Zidovudine.

Further agents for use with the scaffolds described herein include anyone or more of a variety of anti-viral drugs. Anti-viral drugs are wellknown in the art. Illustrative anti-viral agents include, but are notlimited to Abacavir—anti-HIV. NRTI drug. “Ziagen” (ViiV Healthcare). Incombination formulations: “Trizivir” and “Kivexa/Epzicom”,Aciclovir—anti-HSV, Acyclovir, Adefovir, Amantadine, Amprenavir,Ampligen, Arbidol, Atazanavir, Atripla, Boceprevir, Cidofovir, Combivir,Darunavir, Delavirdine, Didanosine, Docosanol, Edoxudine, Efavirenz,Emtricitabine, Enfuvirtide, Entecavir, Entry inhibitors, Famciclovir,Fixed dose combination (antiretroviral), Fomivirsen, Fosamprenavir,Foscarnet, Fosfonet, Fusion inhibitor, Ganciclovir, Ibacitabine,Immunovir, Idoxuridine, Imiquimod, Indinavir, Inosine, Integraseinhibitor, Interferon type III, Interferon type II, Interferon type I,Interferon, Lamivudine, Lopinavir, Loviride, Maraviroc, Moroxydine,Methisazone, Nelfinavir, Nevirapine, Nexavir, Nucleoside analogues,Oseltamivir, Peginterferon alfa-2a, Penciclovir, Peramivir, Pleconaril,Podophyllotoxin, Protease inhibitor, Raltegravir, Reverse transcriptaseinhibitor, Ribavirin, Rimantadine, Ritonavir, Pyramidine, Saquinavir,Stavudine, Synergistic enhancer (antiretroviral), Tea tree oil,Tenofovir, Tenofovir disoproxil, Tipranavir, Trifluridine, Trizivir,Tromantadine, Truvada, Valaciclovir, Valganciclovir, Vicriviroc,Vidarabine, Viramidine, Zalcitabine, Zanamivir, and Zidovudine.

In further examples, the surface chemistry of the scaffold may bealtered. In this regard, the surface may be modified by covalent(direct) attachment of biomolecules or by adsorption of biomolecules.Illustrative biomolecules include any of the biomolecules disclosedherein, such as but not limited to cellular proteins, any of thepolymers described herein, collagens, extracellular matrix components,cytokines, growth factors, anti-inflammatory mediators and others.

In certain examples, the surface structure of the scaffold may bemodified to provide texture, roughness and/or three-dimensionalunevenness to the scaffold. The surface roughness of the scaffold may bealtered by chemical etching or by physical etching. The scaffoldsdescribed herein may also be used as delivery devices for therapeutics,wherein the therapeutic comprises the minced tissue, which may include acombination of cells, extracellular matrix and/or inherent growthfactors. The scaffold portion of the implant may thus permit hormonesand proteins to be released into the surrounding environment.

The fully demineralized layer(s) of the scaffold may have a calciumcontent of, for example, from about 0% to about 2%. The scaffold mayfurther comprise a partially demineralized bone layer between themineralized segment and the demineralized bone segment. A partiallydemineralized bone segment may have a calcium content from about 2% toabout 10% calcium. The partially demineralized segment may besignificantly smaller than either the fully demineralized or themineralized segments.

The bone scaffolds described herein may be used to repair of ligaments,tendons, cartilage or growth plates in the shoulder, hand, elbow, knee,foot, ankle or any other anatomical location as needed. Furthermore, thescaffold of the present invention may be applied to replace or repairany of a variety of joints.

The fully demineralized layer(s) of the bone scaffolds described hereinmay be formed by any appropriate method. In some examples themineralized layer(s) may be masked before demineralization. Any methodor substance may be used to mask the bone that is resistant todemineralization conditions and in some examples, this masked region maybe readily removed after demineralization. Alternatively, the bones maybe masked with a material that is biologically and physiologicallycompatible and therefore does not need to be removed afterdemineralization. In some examples the layer to remain mineralized maybe masked with wax or with a polymer. In one illustrative example, thepolymer is any polymer that may be removed with acetone or ethanol.

Masking may be particularly difficult to achieve precisely with theexacting thicknesses (and ratios of thicknesses, e.g., of mineralized todemineralized) described herein. Thus, in some examples more precisemeans be used, including in particular, starting with bone that includesboth cortical and cancellous bone, as will be described in greaterdetail below.

As mentioned above, the scaffolds described herein may be treated toincrease the strength of the scaffold. For collagenous structures,crosslinking provides greater mechanical strength and a degree ofresistance to proteolytic enzyme degradation, increasing the in vivolifetime of the cancellous bone scaffolds. The cancellous bone scaffoldsmay be crosslinked, either chemically or mechanically. Crosslinking thecancellous bone scaffold may substantially increase the mechanicalintegrity of the scaffold, without substantially altering thecytocompatibility of the scaffold. Additionally, both the physical andchemical crosslinking methods may be biologically compatible.Non-limiting examples of physical crosslinking may be dehydrothermalcrosslinking or crosslinking by exposure to gamma radiation orultraviolet radiation. Physical crosslinking methods of proteinaceousmaterial such as the cancellous bone scaffolds are well known in theart. Alternatively or additionally, the bone scaffold may be chemicallycrosslinked. Functional groups that specifically react with amines maybe, but not limited to, aldehydes, N-hydroxysuccinimide (NHS),isocyanate, epoxide and acrylate. The collagen material of thecancellous bone scaffold is known to comprise lysine residues that maybe crosslinked. Functional groups that are non-selective may be, but notlimited to, active esters, epoxides, azides, carbonylimidazole,nitrophenyl carbonates, tresylate, mesylate, tosylate and isocyanate.Other agents may also be employed for chemically crosslinking thecancellous bone scaffold, including, but not limited to, carbodiimides,genipin, aldehydes such as glutaraldehyde and formaldehyde, acyl azide,poly-epoxy compounds, butanediol diglycidyl ether, dye mediatedphotooxidation or tannic acid. A mixture of crosslinking agents may beused. The choice of crosslinking agent may depend on the amount ofcrosslinking desired, although this may also be controlled bycontrolling the time of the crosslinking reaction and/or by controllingthe concentration of the crosslinking agent.

In some examples, the osteoinductivity of the scaffold (and particularlythe demineralized layer) may be removed from the cancellous bonescaffold, e.g., using peroxide or other agents, as described above.

The scaffold described herein may be useful in treating injuriesinvolving interfaces within connective tissues. The major applicationsinclude repair of ligaments, tendons, and cartilage. Ligaments are densebands of connective tissue composed primarily of type I collagen thatconnect bones to other bones. Ligaments function as motion guides andjoint motion restrictors. At all articulating joints (neck, spine,shoulder, elbow, wrist, hip, knee, ankle) in the body, these tissues areplaced under constant dynamic loading. An injury known as a sprainresults when the ligaments are stretched, and in some cases, stretchedseverely enough to be torn. While in some cases, ligament tears can healon their own, other cases show a lack of inherent healing capacity. Ifleft untreated or if treated improperly, ligament tears can lead tochronic disability including arthritis at the affected joint. Tendons,like ligaments, are dense collagenous tissues found at everyarticulating joint in the body. Tendons, however, connect muscles tobone, allowing the force produced by the muscles to be translated intomotion. When overloaded, tendons are at risk for tearing and in somecases require surgical replacement to return joint motion and preventmuscle atrophy.

FIGS. 2A-2D illustrate examples of grafts as described herein includinga mineralized layer 103 and a demineralized layer 101. As mentionedabove, in some examples the scaffold may have dimensions that areoptimized to prevent breaking, improve delivery and enhance integrationinto the tissues of the body. For example, in some examples the scaffold100 may be configured as rectanguloid body having dimensions in whichthe length 107 is between about 15 and 50 mm (e.g., between 15-35 mm,between 15-30 mm, between 20-30 mm, between 20-28 mm, between 15-25 mm,etc.), the width 105 is between about 10-26 mm (e.g., between 12-18 mm,between 15-20 mm, between 10-15 mm, between 13-17 mm, etc.), and thethickness is between 2-6.5 mm (e.g., between 2-6.0 mm, between 2-5.5 mm,etc.).

The thickness of the demineralized 111 and mineralized 109 layers may bedifferent. For example, as shown in FIG. 2B, the demineralized layer111′ is thicker (e.g., between 60-99% of the thickness of the body ofthe scaffold, e.g., between 60-95%, between 65-99%, between 70-95%,etc.). The thickness of the mineralized layer 109′ may be, e.g., betweenabout 40 and 1% of the thickness of the body (e.g., between 35-1%,between 35-1%, between 35-5%, etc.). In particular, the thickness of themineralized layer may be limited to a maximum (regardless of thethickness of the demineralized layer), such as about 1.5 mm or less(e.g., 1.4 mm or less, 1.3 mm or less, 1.2 mm or less, 1.1 mm or less,1.0 mm or less, 0.9 mm or less, 0.8 mm or less, 0.7 mm or less, betweenabout 1.4 mm and 0.1 mm, between about 1 mm and 0.1 mm, etc.)

Alternatively, in some examples, as shown in FIG. 2C the mineralizedlayer 109″ is thicker (e.g., between 60-90% of the thickness of the bodyof the scaffold, e.g., between 60-85%, between 65-85%, between 70-80%,etc.). The thickness of the demineralized layer 111″ may be, e.g.,between about 40 and 10% of the thickness of the body (e.g., between40-15%, between 35-10%, between 35-15%, etc.).

In some examples the graft may be configured for insertion into thebody. For example, the graft may include a thin mineralized bone layerformed adjacent to a thicker demineralized layer to provide bendingwithout breaking the mineralized layer.

In some examples, the graft may include regions of fully demineralizedbone. FIG. 2D illustrates one example of a scaffold including regionsbetween mineralized layer regions formed by demineralizing the bone inthese regions. These regions may be one continuous region (e.g., alongitudinally-extending region, etc.) or a plurality of separateregions. In FIG. 2D, the scaffold 100 includes a central longitudinalregion 121 through the mineralized layer (the longitudinal axis may bereferred to as the long axis and may line in the direction of the length107 of the scaffold). The demineralized bending region may providesufficient flexibility so that the mineralized bone layer 103, 103′ oneither side of the region may flex or bend for inserting into a cannulato be delivered into the body. FIG. 2E shows another example of ascaffold implant that includes a plurality of demineralized regions 121,121′, 121″ formed along the length of the mineralized bone layer 103.The demineralized regions (bending regions) may extend completely to thedemineralized layer 101 or partially to the demineralized layer.

In some examples the scaffold may be notched to allow it to bend, e.g.,by notching the mineralized side (not shown). This may be done inaddition to or instead of including a region of demineralized bone.

Any of the scaffolds described herein may include one or more channelsor openings configured to receive suture for attaching the device to thebody and/or for attaching the tissue (connective tissue) to thescaffold. In particular, the openings or channels may be suture channelsthat may extend in some examples transversely through the thickness, asshown in FIG. 3 . In FIG. 3 two suture channels 304, 306 are shownthrough the middle region of the scaffold 300; these channels may passcompletely through the mineralized layer 103 and in some cases throughthe demineralized layer 101. Alternatively in some examples a suturechannel may extend just partially into the thickness and then traveltransversely (not shown) and then exit the same side it entered from ora different side.

In any of these examples the suture channel may be treated to preventsnagging, treating or breaking the suture (e.g., on the porous structureof the bone) or risk damaging the bone. For example, the bone regionsforming the channel may be compressed, polished, and/or sealed toprevent access to the trabecula within the layer(s). In some examplesthe opening into the scaffold may be rounded to remove any sharp edges.

Any number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.) of openings into thebone layers may be formed in the scaffold. The positions of the one ormore openings may be arranged in any appropriate region of the scaffold,such sat the central region, an upper region 308, a lower region 310, aright side region 312 or a left side region 314, and/or any combinationof these. In some examples the scaffolds may be pre-threaded with one ormore sutures through the suture openings, and provided the user (e.g.,doctor) for implantation.

Any of the scaffolds described herein may include rounded or bevelededges. For example, a scaffold may include edges (outer edges of thebody) that are rounded, e.g., having a radius of curvature of betweenabout 0.1 mm and 2 mm (e.g., between about 0.2 and 2 mm, between about0.3 and 2 mm, between about 0.1 and 1.5 mm, between 0.1 about 1 mm). Forexample, FIG. 4A shows one example of a scaffold having rounded edges.FIG. 4B is an example of a scaffold having tapered edges. In FIG. 4A,the rounded edges 406 extend around the perimeter of the scaffold, onboth the demineralized 401 and mineralized 403 layers of the scaffold,which may prevent trauma to the tissue into which the scaffold isinserted. In some examples just the demineralized (e.g., outward-facing)portion of the scaffold includes rounded and/or tapered edges. FIG. 4Bshows an example of a scaffold 400 including tapered edges 416.

In some examples the scaffold may be formed of a single portion of abone that is treated to form the demineralized and mineralized layers inan arrangement as described herein. In some examples the scaffold layersmay be separately formed and combined either before or during insertioninto the patient's body. For example, in some examples the demineralizedlayer may be formed as a single layer that is mated with the mineralizedlayer. The demineralized layer may be attached (affixed) to themineralized layer. For example the mineralized and demineralized layersmay be chemically, mechanically, thermally or otherwise bonded together.For example in some examples a chemical adhesive may be used to bond thedemineralized and mineralized layers together. In some examples themineralized and demineralized layers may be shaped to interlock ofengage in a predefined manner. For example the demineralized layer mayinclude a projection into which the mineralized layer inserts, or viceversa (or both).

FIG. 5A illustrates one example of a scaffold formed of initiallyseparate demineralized 501 and mineralized 503 layers. In FIG. 5B, theinitially separate layers may be coupled together by a suture 511 orsutures 511′, that may pass through pre-formed suture openings orpassages 512. Thus, the suture may be used to affix the two (or more)layers together during a procedure to insert/apply them. The differentlayers may be formed of different regions of bone and/or differentregions of the same or different bone types (e.g., cancellous andcortical, etc.).

Also described herein are scaffolds that are cancellous and corticalbone scaffolds.

For example, a section of bone comprising both cancellous and corticalbone regions may be used as a starting material that may be treated todemineralize the bone. The demineralization rate and required conditionsfor demineralization of cancellous and cortical bone are different. Atmultiple points in the body (i.e. the head of long bones) there is arecortical cancellous interfaces. Such interfaces may be used to formcortical and cancellous scaffolds from a single allograft material(e.g., without requiring a lamination step, such as shown in FIGS.5A-5B) or other attachment of two disparate bone types. The porestructure of cancellous bone material may allow for better penetrationof the demineralization solution and an overall higher surface area tovolume ratio enabling the demineralization of this portion of the bonemore effectively. This change in porosity and resistance todemineralization could be used in allograft material to form a scaffoldwith a mineralized side and a demineralized side. For example, the wholebone may be placed in a demineralization solution for an amount of timesufficient to render the cancellous side to the appropriate level ofdemineralization (e.g., <8% Calcium, <7% Calcium, <6% Calcium, <5%Calcium, <4% calcium, <3% calcium, <2% calcium, etc.). The bone scaffoldmay then be placed with the cancellous side in demineralization solutionand the cortical side above the level of solution. The reduced porosityof the cortical bone may prevent the demineralization solution fromwicking through the bone as it does in cancellous bone. Either before orafter the demineralization, the bone material may be trimmed or cute tothe appropriate dimensions as described herein.

The resulting implant may therefore be a scaffold having a first layerthat is formed of a demineralized cortical bone that is immediatelyadjacent (and in some examples contiguous with, e.g., formed of aunitary structure) a mineralized cortical layer of bone.

Any of the implants described herein may be hydrated or non-hydrated,for storage prior to use. Before implantation into the body, they maybehydrated, e.g., by soaking or adding a fluid, e.g., water, saline,buffer, protein solution (such as platelet-rich plasma, PRP), etc.

Thus, any of the scaffolds described herein may include a trapezoidal(e.g., rectanguloid) body that may be, e.g., wider on a superior side,narrower on an inferior side. Alternatively, in some examples thescaffold may be oval shaped (or may have an oval cross-section, e.g.,having rounded edges). The scaffold may be formed of any appropriatesource of bone (e.g., cancellous, cortical, both cancellous andcortical, etc.) from any appropriate region (e.g., pelvis, etc.). Thedensity of the bone may be different between the mineralized anddemineralized regions/layers; thus the size and/or number oftrabeculae/porosity may be different between the mineralized anddemineralized bone layers.

In some examples all or some of the surfaces of the mineralized and/ordemineralized layers may be grooved or textured. For example, just theinferior side may be grooved or just the superior surface of bothmineralized and demineralized or just mineralized or just demineralizedlayers. Other textures may be used, including grids, parallel channels,etc.

As mentioned above, any appropriate method of making the scaffold may beused, including creating separate mineralized and demineralized layersusing wicking (e.g., pressure-limited or driven wicking) topredetermined and precisely controlled thicknesses. One or more maskingagents may be used, including masking agents having selected viscositiesor densities, and/or controlling temperature (decrease temperature orincrease temperature) of the masking agent and/or demineralizingsolution to control the depth of demineralization compared to theremaining mineralized layer.

The scaffolds described herein may be sterilized and may be configuredfor sterilization. For example, these scaffolds may be sterilized bygamma sterilization, e-beam sterilization, supercritical carbon dioxidesterilization, etc.

In use, these scaffolds may be implanted into a patient in need thereofin any appropriate technique. In particular, these scaffolds may beimplanted via an arthroscopic procedure, a mini-open procedure, etc. Asmentioned, the scaffold may be configured for insertion to the body viaa cannula, and may be affixed via a suture. In some examples thescaffold may be used to repair tendon tears on the articular surface; insome examples the scaffold may be used to repair tendon tears on thebursal surface.

Any appropriate tissue may be repaired, including, but not limited torotator cuff tissue. For example, FIGS. 6 (1)-6(10) illustrate onemethod of using a scaffold as described herein to repair a rotator cuff.In FIG. 6 (1), the tissue to be repaired may be prepared, e.g., byremoving unwanted tissue and/or debriding. In FIG. 6 (2), the boneyregion to which the scaffold will be anchored is prepared, by applyingone or more anchors (e.g., medial anchors) including sutures(“threads”). A bone shaver may be used to cut away the region into whichthe scaffold will be applied, as shown in FIG. 6 (3). The mineralizedside may be placed down onto the exposed inner bone region, so that themarrow may be driven, e.g., by pressure, into the trabecula of themineralized bone helping to vascularize the scaffold to the native bonetissue. In FIG. 6 (4), the scaffold (which may be preloaded on thesutures, e.g., via one or more suture channels, as described above, maybe slid down through a cannula (not shown) and into the bone. In FIG. 6(5), the scaffold is applied onto the prepared bone and may be anchored,via the suture, in place, as shown in FIG. 6 (6). In FIG. 6 (7), thetendon to be repaired may be sutured with the same sutures used toanchor the scaffold (or coupled to the scaffold anchors) and may bepulled down onto the scaffold outer surface (e.g., the demineralizedlayer of the scaffold). FIG. 6 (8) shows the tendon fully covering thescaffold, and in FIG. 6 (9) and FIG. 6 (10) the sutures may be used toanchor and secure the tendon to the scaffold. In some examplesadditional material may be used to secure the tendon to the scaffold. Insome examples, no additional securement or augmentation is notnecessary.

Stiffness

As mentioned above the implants (e.g., grafts) described herein may beconfigured to be sufficiently flexible so that they may be insertedthrough a cannula without requiring a specific hinge region. Forexample, any of the bone scaffold grafts described herein may beconfigured so that they may be bent (e.g., folded, curled, etc.) withoutbreaking or cracking. This may be achieved, at least in part, bycontrolling the overall thickness of the mineralized portion, and/or therelative thickness of the mineralized vs. the demineralize layers. Forexample, the bone scaffold grafts described herein may have an overallgraft thickness that is limited to between 4.5-6.5 mm thick or less(e.g., 6.5 mm or less, 6.0 mm or less, 5.5 mm or less, 5.4 mm or less,5.3 mm or less, 5.2 mm or less, 5.1 mm or less, 5.0 mm or less, 4.9 mmor less, 4.8 m or less, 4.7 mm or less, 4.6 mm or less, 4.5 mm or less,etc.) in which the maxim thickness of the mineralized portion is about1.5 mm or less (e.g., 1.4 mm or less, 1.3 mm or less, 1.2 mm or less,1.15 mm or less, 1.1 mm or less, 1.05 mm or less, 1.0 mm or less, 0.95mm or less, 0.9 mm or less, 0.8 mm or less, etc. between about 100 μmand 1.25 mm, between about 100 μm and 1.1 mm, between about 150 μm and1.1 mm, between about 150 μm and 1.0 mm, etc.).

Surprisingly, for bone scaffold grafts as described herein, in which thegraft is formed of adjacent mineralized/demineralized layers in whichthe mineralized layers is about 1.5 mm or less, typically where thethickness of the bone scaffold grafts is between about 4.5 and 6.5 mmthick, the bone scaffold grafts is sufficiently flexible that, whenhydrated, it may be easily bent without breaking. For example, thestiffness of bending a sheet-like bone scaffold graft (e.g., a graftformed into a planar sheet in which one side is the mineralized layerand the other is the demineralized layer) may have a stiffness, whenmeasured as mean bending stiffness, that may be less than about 1millinewtons meters (mNm) (e.g., less than about 1.2 mNm, less thanabout 1.1 mNm, less than about 0.9 mNm, less than about 0.85 mNm, lessthan about 0.8 mNm, less than about 0.75 mNm, less than about 0.7 mNm,less than about 0.65 mNm, less than about 0.5 mNm, less than about 0.25mNm, less than about 0.1 mNm, etc.). Bone scaffold grafts having athickness of the mineralized layer that is greater than 1.5 mm typicallyhave a mean bending stiffness that is greater than this, and may bedifficult to bend without cracking, breaking or otherwise damaging thebone scaffold graft.

Thus, described herein are bone scaffold grafts that have a maximumthickness that is about 1.5 mm or less (e.g., 1.4 mm or less, 1.3 mm orless, 1.2 mm or less, 1.15 mm or less, 1.1 mm or less, 1.05 mm or less,1.0 mm or less, etc.). As mentioned, the overall thickness of the graftmay be, e.g., between about 4.5 and 6.5 mm or less, with the majority ofthe thickness of the graft being formed of demineralized bone material.For example, the demineralized layer may be greater than 65% of thethickness of the bone scaffold graft (e.g., greater than 67%, greaterthan 70%, greater than 75%, greater than 80%, greater than 85%, etc.).These highly flexible bone scaffold graft implants described hereinhaving greater than 60% or more of the thickness being demineralizedbone (and having a maximum mineralized layer thickness of 1.5 mm orless, e.g., 1.4 mm or less, etc.) may also be easier to cut, withoutcracking or damaging the graft, than implants having higher thicknessesof the mineralized layer. Cutting to size may be important forimplanting the graft material into the patient, as this may allowcustomization of the insertion/implantation site.

In addition to the enhanced flexibility, e.g., low mean bendingstiffness of less than about 1 mNm, which may allow the bone scaffoldgrafts described herein (e.g., having a maximum thickness of themineralized layer of about 1.5 mm of less) to be easily bent and cut toshape/size, any of the bone scaffold grafts described herein may also beoptimized for conformability, allowing the graft material to conform toa potentially irregular surface, such as the prepared bone surface ontowhich the implant is to be applied. The prepared surface may be flat oruneven (e.g., not flat). Outcomes are expected to be far better if thebone scaffold graft sits flush against the bone onto which it is beinggrafted, even when the bone surface is curved or irregular. Traditional,more rigid or stiffer bone scaffold grafts are not capable of beingapplied flush against such uneven surfaces. Typically, the bone scaffoldgrafts described herein may be highly conformable.

This conformability may be a function of both the high flexibilitydescribed above, as well as a relatively high compressibility of thebone scaffold grafts described herein. For example, the bone scaffoldgrafts described herein, formed as adjacent sheets ofmineralized/demineralized material, may be relatively highlycompressible as a function of the thickness of the demineralized portionand/or the porosity of the graft material, particularly the porosity ofthe demineralized layer. The hydrated graft, ready for implantation, maybe both highly conformable, as described above, and highly compressible,particularly where greater than 60% (e.g., greater than 65%, greaterthan 70%, greater than 75%, greater than 80%, greater than 85%, etc.) ofthe thickness of the graft is demineralized. In some examples a grafthaving greater than 66% (e.g., greater than 67%, greater than 70%,greater than 75%, greater than 80%, greater than 85%, etc.) of thethickness being formed of demineralized bone material, with anuncompressed thickness of between about 6.5 and 4.5 mm, may becompressed to a thickness of about 2 mm or less, when a force normal tothe layer(s) is applied.

As mentioned above, the porosity of the bone scaffold grafts may beselected so that these grafts have a compressibility and/or flexibility(e.g., stiffness) as described. For example, the material used to formthe bone scaffold grafts described herein may be selected to have aporosity so that the density of the material (where density may be aproxy for the porosity) is less than about 3.0e-4 g/mm³ (e.g., about2.9e-4 g/mm³ or less, about 2.8e-4 g/mm³ or less, about 2.7e-4 g/mm³ orless, about 2.6e-4 g/mm³ or less, about 2.5e-4 g/mm³ or less, 2.4e-4g/mm³ or less, 2.3-4 g/mm³ or less, 2.2e-4 g/mm³ or less, 2.1e-4 g/mm³or less, 2.0e-4 g/mm³ or less, 1.9e-4 g/mm³ or less, etc.). Inparticular, the density may be 2.5e-4 g/mm³ or less (e.g., 0.00025 g/mm³or less). This density may be maintained by the mineralized region andapproximately by the demineralized region, following formation. Thisrange of porosity may also be important, in combination with thethickness of the mineralized region described above, for achieving thehighly flexible grafts (e.g., low mean bending stiffness) graftsdescribed herein. In some examples the porosity may also besubstantially homogeneous.

Also described herein are bone scaffold grafts having a higher stiffnessthan those described herein, but in which the dimensions arenarrower/smaller (e.g., having length and/or width dimensions that are10 mm or less (e.g., 8 mm or less, 7 mm or less, 6 mm or less, 5 mm orless, etc.). Smaller bone scaffold grafts (e.g., having at least twodimensions (e.g., width and thickness) that are less than x mm (e.g.,where x is 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, etc.) may bestiffer than those described herein. However, in some examples, smallerbone scaffold grafts may have the mean bending stiffness,compressibility and porosity as described above (e.g., a mean bendingstiffness of 1 mNm or less, a density of less than 2.6e-4 g/mm³, etc.)

In general, the bone scaffold grafts described herein may be provided inany length and width, and may be cut down for implantation by thephysician. For example, the bone scaffold grafts may have dimensions(length×width) of between 50-10 mm by 80-20 mm, e.g., 25 mm×50 mm.

The thickness of the mineralized layer of bone scaffold graft may varyby less than +/−10% (e.g., less than +/−9%, less than +/−8%, less than+/−7%, less than +/−6%, less than +/−5%, less than +/−4%, less than+/−3%, less than +/−2%, less than +/−1%, etc.). This high level ofuniformity in the thickness of the mineralized (and conversely thedemineralized) layer(s) may provide uniformity in the key properties,such as stiffness, compressibility and compliance, discussed above,which may not otherwise be possible.

The bone scaffold grafts described herein may also or additionallyadvantageously be free of blood proteins. As described above, grafts maybe provided dehydrated and may be rehydrated prior to use. The graftsmay be rehydrated prior to cutting down to size before implantation.

Methods of Forming Bone Scaffold

The bone scaffold grafts described herein may be formed by anyappropriate technique. In particular, these bone scaffold grafts may beformed by starting with a material that is cut from donor bone materialto dimensions larger than those described above, typically by awhole-number multiple, but at the target thickness (e.g., less thanabout 5.5 to 4.5 mm) so that the bone may later be cut to the final sizeafter washing. For example, the material may be taken from donor femur,tibia, and humerii. In particular, the material may be taken from thehead region of the bone (as there may be less suitable cancellous bonein the stems of the bones). The bone donor material may be taken fromolder donors, and in particular donors having lower bone density.

The bone may then be washed, e.g., with a detergent, to remove anyorganic material. Bone material may be pre-screened, either before orafter washing), to confirm that the bone is sufficiently porous; thedensity may be used to confirm porosity. The porosity may also beconfirmed as substantially uniform. Optionally, the cut bone may berinsed and further dissected into the desired sizes (e.g., “finedissection” to a length of between about 50 mm to 10 mm, width ofbetween about 80 mm to 20 mm, and a thickness of between about 4.5 mm to5.5 mm or less, e.g., between about 5.5 mm and about 2 mm). Followingthis fine dissection step, the bone sections may be defatted, e.g., bywashing in a defatting agent, such as acetone, then dried. Alternativelythe fine dissection step may be performed after the masking anddemineralization steps, described below.

The bone may then be masked to allow demineralization of the desiredthickness (typically greater than 65% of the thickness). As describedherein, the cleaned and defatted bone may have a thickness of less thanabout 5.5 mm (e.g., less than 5 mm, less than 4.5 mm, etc.). The methodsdescribed herein may permit less than 1.5 mm (e.g., less than 1.4 mm,less than 1.3 mm, less than 1.2 mm, less than 1.1 mm, etc.) to be remainunmasked.

The masking agent may be a liquid that is applied (coated, brushed,dipped, etc.) to just one side (the mineralized side) to protect it fromthe demineralization treatment. Because of the relative importance ofthe thickness of the mineralization as discussed above, e.g., inproviding the flexibility, compressibility and overall compliance of thebone scaffold graft, the masking material should be chosen to havespecific properties, described in greater detail herein. Once themasking agent is applied and dried, the masked bone material may bedemineralized, e.g., by treating with a bone demineralizing agent, suchas HCl, then buffered and rinsed. The bone material may then bede-masked by an agent (e.g., acetone) to remove the masking material,rinsed, and prepared for storage, either dry or wet storage.

The masking agent appropriate for masking thickness of the porous bonemust be able to controllably integrate to just the thickness of the bonestructure desired, typically a thickness of less than 1.5 mm (e.g., lessthan 1.4 mm, less than 1.3 mm, less than 1.2 mm, less than 1.1 mm, lessthan 1.0 mm, less than 0.9 mm, less than 0.8 mm, less than 0.7 mm,between 5.5 and 5 um, between 5.5 and 0.01 mm, between 5.5 and 0.1 mm,etc.). Preferably, the masking agent is a liquid material (e.g., atprocessing temperature, such as room temperature or cooler). Thus, themasking agent must have a low vapor pressure (e.g., must not bevolatile) so that the material does not invade, e.g., blooming deeperinto the thickness of the porous bone. The masking agent must also havea relatively high viscosity, so that the liquid masking agent doespenetrate into the desired thickness (e.g., up to 1 mm, etc.). Themasking agent may also have a relatively rapid cure time (e.g., curingnearly completely by within 2 hours, 3 hours, 4 hours, 5 hours, 6 hours,7 hours, 12 hours, 15 hours, 20 hours, 24 hours, etc.). Further, thecuring process should not be exothermic, or should be only mildlyexothermic (less than a few degrees C.), so as not to damage the bonematerial. Finally, the masking agent material should be readily andthoroughly removable by a processing step (e.g., acetone).

In some examples the masking material may be a cyanoacrylate. Howevernot all cyanoacrylates work. Many cyanoacrylates have a relatively lowviscosity and low vapor pressure (e.g., are volatile). For example,ethyl cyanoacrylates, butyl cyanoacrylates, and methyl cyanoacrylatestypically have a very low vapor pressure and readily vaporize, spreadingfurther within the pores of the bone than the desired thickness. Incontrast, cyanoacrylates having intermediate-length, polar side chainssuch as ethoxyethyl cyanoacrylates, may work surprisingly well. Incontrast, cyanoacrylates having longer side chains (such as octylcyanoacrylate) do not work well; although they are less volatile, theydo not cure within a reasonable time period.

Thus, an appropriate masking agent may include a material including acyanoacrylate having a polar side chain (R group) including an oxygen(e.g., an ether group). In particular, the masking agent may include anethoxyethyl cyanoacrylate. Other examples may include methoxyproplycyanoacrylate and ethoxymethyl cyanoacrylate. These cyanoacrylates mayhave a low blooming (little vapor) and be relatively quick curing,permitting accurate masking.

Any of the masking materials described herein (including those havingcyanoacrylates with a polar side chain (R group) including an oxygen (anether containing side chain, such as ehtyoxyethyl cyanoacrylate) mayinclude a thickener to reduce the viscosity. The thickener may be apolymer compatible with the cyanoacrylate, such as polymethylmethacrylate (PMMA). In particular, it was found that lower molecularweight, relatively higher percentages of PMMAs worked better than largermolecular weight PMMA, including at lower percentages. For example, aPMMA thickener having an average molecular weight of about 350,000 orless may be used at between about 10% and 4% (e.g., about 8%) to adjustthe viscosity of the masking material.

The masking agent may be removed with acetone, methyl ethyl ketone,nitromethane, or methylene chloride. In particular, the masking agentmay be removed from the graft following demineralization using acetone.

In use, the masking agent (also referred to herein as a maskingmaterial) may be applied to the bone after cleaning; the application isconfigure so that only the narrow range (e.g., less than 1.5 mm thick,flat/planar side) of porous bone is masked. Masking material may beapplied in any manner that allows precise control of the depth ofmasking, e.g., to limit to the thickness (1.5 mm or less, 1.4 mm orless, 1.3 mm or less, 1.2 mm or less, 1.1 mm or less, 1.0 mm or less,0.9 mm or less, 0.8 mm or less, etc.). The depth may also be consistentacross the length and width of the bone graft. Finally, the method forapplying the masking material may be highly reproducible.

Examples of methods of masking the bone scaffold graft material mayinclude stamping, dipping, painting, spraying, etc. The maskingprocedure may be performed in a temperature and/or moisture-controlled(e.g., humidity controlled) environment or chamber. The temperature maybe at room temperature in some examples.

For example, in some examples the bone scaffold graft material may becleaned, then dipped into the masking agent; the masking agent may beheld within a bath, e.g., a small, shallow chamber, into which theporous surface to be masked is dipped to the depth to be masked (e.g.,less than 1.5 mm, less than 1.0 mm, etc.). The bone scaffold graftmaterial may be held in the pool for sufficient time so that the maskingagent invades the pores (e.g., 1-30 seconds, 5-20 seconds, 5-15 seconds,5-10 seconds); then removed from the masking agent pool and the maskingagent may be allowed to cure. In some examples, the bone scaffold graftmaterial may be allowed to cure inverted, to further allow the bonegraft material to penetrate to the controlled depth. During curing(drying) the graft may be moved one or more times, which may helpprevent the masking agent from adhering the bone material to thesurrounding surfaces.

In general, the masking agent, and therefore the resulting thin layer ofmineralized bone, may be consistent in its width across the bonescaffold graft. As discussed above, the thickness of the mineralizedlayer of bone scaffold material across the graft may vary by less than+/−10% (e.g., less than +/−9%, less than +/−8%, less than +/−7%, lessthan +/−6%, less than +/−5%, less than +/−4%, less than +/−3%, less than+/−2%, less than +/−1%, etc.). The methods for fabrication describedherein, including the composition of the masking agent, may provide fora high level of uniformity in the thickness of the mineralized (andconversely the demineralized) layer(s). This may also provide uniformityin the critical properties, such as stiffness, compressibility andcompliance, discussed above.

In particular, the ability of the masking agent to cure quickly (e.g.,faster than 1 hour), and have sufficient viscosity to coat the porousbone without being wicked to deeply into the bone material, as well asthe low vapor release may allow for precise control and consistency inthe masking and therefore formation of the thin mineralized layer.

As discussed above, the bone material may be cleaned and defatted priorto masking. The defatting step may include the use of a protease, whichmay be used before, after or concurrent with acetone/ethanol. Inparticular, a protease (e.g., a detergent including protease) may be useas part of the defatting step, which may use acetone and/or ethanol. Thedefatting step may help in the masking agent to coat and protect theregion of the bone to remain mineralized. The acetone may be washed off.The protease may also remove the discoloration of the bone material dueto blood. The defatting step removes organic material (not limited tofat).

FIGS. 7A-7C illustrate examples of grafts as described herein. Eachgraft shown is approximately 25 mm by 15 mm and is formed fromcancellous bone. In FIG. 7A the demineralized side 701 of the graft isshown. FIG. 7B shows a mineralized side 703 of the graft. FIG. 7C showsa side view of a graft such as the one shown in FIG. 7A, showing themineralized layer 703 immediately adjacent to the demineralized layer701. As described above, the mineralized layer is less than about 1 mmin this example. In FIG. 7C the thickness of the graft is approximately4.5, and the thickness of the calcified region is approximately 1.5 mm.This graft is flexible and compressible, as described herein (notshown).

FIG. 7D illustrates the flexibility of the graft shown in FIG. 7C. Inthis example the graft has been folded over itself completely, as shown.The graft, including in particular the mineralized layer, does not crackor become damaged despite bending relatively easily, and will return toits original flat shape following release of the bending force.Similarly, the same graft is also compressible and conforms to thesurfaces applying the compression, as shown in FIGS. 7E and 7F. In thisexample the graft is shown held between two fingers (FIG. 7E) and isfurther shown compressed between these two fingers in FIG. 7F.

EXAMPLE

FIG. 8 illustrates one example of a method 800 as described herein. Thismethod uses donor bone tissue to from the bone matrix grafts describedherein. Initially, a gross dissection may be performed 801 from thedonor bone material. For example, the donor bone tissue may be cut intoslices having a thickness of between about 5.5 and 3.5 mm. The lengthand width may vary, and may depend upon the size of the region of donorbone tissue from which the bone is being removed. For example, thinpieces of bone may be taken from donor femurs, tibias, and/or humerii,and in particular from the head regions of the bones, as there may beless suitable cancellous bone in the stem regions of the bones. Althoughother thicknesses may be used (e.g., between 10 mm and 2 mm), athickness of between about 5.5 mm and 3.5 mm may be particularly wellsuited for use in rotator cuff repair. The cut material may be weighedand measured, from which an approximation of the density (and thereforeporosity) of the bone may be made.

The cut donor bone tissue may then be washed 803. For example, the cutdonor bone slices may be washed in a protease detergent (e.g., a 2%Prolystica solution), which, in some examples, may include a combinationof a protease and a detergent/surfactant, to remove organic material.Multiple was steps may be performed, such as washing for 3 or morecycles. In some examples each cycle of washing may include at least 10mL of washing solution per gram of bone material. Washing may be donewith agitation (e.g., rotating at approximately 150 rpm) for about 1hour per wash cycle. Fresh protease detergent may be used with eachcycle, replacing the used detergent between each wash. A final wash maybe performed for a longer time, e.g., overnight (˜10-15 hrs), forexample, using 15 mL/g and 150 rpm. Washing may be performed until thecolor (e.g., red color) has cleared.

The sliced bone material may then be rinsed, e.g., in water. Forexample, the thin slices may be rinsed in sterile water for 2 or morecycles using at least 10 mL water per gram of the bone material, withagitation (e.g., 150 rpm) for 30 minutes each. In some examples the bonemay be continuously rinsed. Alternatively, the rinse water may becontinuously replaced, with fresh water, between each rinse. Rinsing maycontinue until the water is no longer sudsy from removed detergent.

The sliced bone may then optionally be more finely dissected 805. Forexample, the thin slices of washed (and defatted) bone material may becut into standard sized strips (e.g., 25 mm by 50 mm) all having thesame thickness (of, e.g., 4.5 mm thick). These rectanguloid-shaped bonescaffold grafts may also be examined manually or automatically toconfirm that the porosity is within the desired range, e.g., based onthe density or on an optical determination of the porosity. Theinspection may accept or reject grafts based on the porosity/densityand/or based on the uniformity of the material. Graft material havingareas of the bone that are within the desired porosity (e.g., not toclose to cortical bone such that material is too dense, and no areas ofvery large pores that would not have adequate structural integrity), andthat are consistent visually with respect to porosity/average pore sizemay be retained. The more fine dissection may additionally oralternatively be performed after demineralization and/or after removalof the masking agent, as described below. For example, the material maybe cut (finely dissected) at the end of the process 805′, as shown inFIG. 8 .

The fine dissection may be performed in stages, so that the length(e.g., of 15.5+/−0.5 mm strips) may be cut first (and inspected betweencuts, or to guide cuts) followed by cutting into widths of a finaldimension, such as about 25.5+/−0.5 mm. These final cut bone scaffoldgrafts may then be provided as ‘blanks’ (e.g., dimensions of 25.5+/−0.5mm, 15.5+/−0.5 mm, and 4.5+1.0 mm in some examples).

An additional washing step (e.g., defatting) step may be performed 807.For example, the ‘blanks’ may be washed in acetone, e.g., for 2 hours ata concentration of 15 mL of acetone per gram of blanks at 150 rpm.Following this, the bone graft material may be washed, e.g., for 3additional cycles, at 10 mL/g for 1 hour at 150 rpm. Replace with freshacetone in between each wash. This step may be coincident with anovernight or longer drying step, to align processing times with ends ofdays. Exact required time between end of defatting and off gassing ofacetone prior to masking has not been quantified). The material may bewashed until the washing acetone is no longer discolored (yellow tint)from defatting.

The bone scaffold graft material may then be masked 809, to define themineralized and demineralized regions 809. In particular, the graftmaterial may be masked so that a thin (e.g., 1.5 mm or less, 1.4 mm orless, 1.3 mm or less, 1.2 mm or less, 1.1 mm or less, 1.0 mm or less,0.9 mm or less, etc.) layer is protect to remain mineralized, allowingthe rest of the graft material to be demineralized. In one example, abath (e.g., a dish) may be prepared with a pool of the masking agent. Inone example the masking agent may be applied (e.g., by syringe todispense the cyanoacrylate mixture) into the bath, creating a shallowlayer (e.g., −1 mm deep or so) in which the cut bone scaffold graftmaterial may be applied. The bath/dish may be agitated, e.g., byrotation, to achieve consistent coverage of the masking agent over theentire surface of the bath/dish. The bone scaffold graft material maythen be placed into the bath/dish on top of the masking agent. Forexample, a sterile plastic forceps may be used to place the ‘blanks’ ofbone scaffold graft material on top of the masking agent. The blanks maysink into the cyanoacrylate until they contact the bottom of thebath/dish (e.g., about 5 seconds), and they may then be removed beforethey can adhere to the bottom surface of the bath/dish. The process maybe repeated with additional blanks, refilling the masking agent, asnecessary. The masked blanks may then be dried 811, e.g., on a dryingrack, masked side down. While drying, the grafts may be moved e.g.,every −5 minutes, such that the cyanoacrylate does not bind the graft tothe drying rack. The bone scaffold graft material may be allowed to dry,e.g., for a total of 1 hr. The blanks (bone scaffold graft material) maybe weighed again after the masking agent has dried, to calculate howmuch cyanoacrylate was absorbed by the blanks.

In some examples, masking may utilize an apparatus, such as a dippingapparatus. An apparatus may include, e.g., magnetic fixation with aplate that contacts the superior/non-masked face, while magnetic studsare placed on the inferior/masked face, thereby holding the bone slicein place. When the bone is dipped into the CA, the magnetic studs maycontact the bottom of the dipping chamber (e.g., dish); this may preventthe graft from having irregular surface contact with the bottom of thechamber, and may also aid in ensuring a reliable and repeatable depthinto which the bone is dipped.

Once dried, the material may be demineralized 813. For example, themasked blanks may be washed for 2 cycles in 0.6 N hydrochloric acid(HCl) at a concentration of 20 mL HCl per gram of bone content of theblanks (not including the masked weights), agitating at 150 rpm for 2hours each cycle. Following the demineralization step the graft materialmay be rinsed, e.g., in a buffer solution. For example, a buffer rinsemay include rinsing the demineralized blanks in neutral (7.0 pH)phosphate buffer solution for 30 minutes with agitation (e.g., at 150rpm) and with at least about 20 mL per gram bone content. The rinse maybe repeated (e.g., rinse again at 20 mL/g and 150 rpm with sterile waterfor 5 minutes).

In some examples, the fine dissection step may be performed aftermasking and demineralization, e.g., after the bone is finishedprocessing. The bone may be manually selected, and grafts cut accordingto the homogenous regions of the bone.

Finally, the demineralized bone scaffold graft material may be de-maskedto remove the masking material from the protected thin layer ofmineralized bone and rinsed 815. For example, the demineralized blanksmay be washed in acetone for 2 cycles at 150 rpm at a concentration of 1L acetone per gram of cyanoacrylate absorbed, for 2 hours each cycle. Asmentioned, in some examples the additional (“fine” dissection) may bedone at this stage, instead of or in addition to before the masking anddemineralization steps.

The grafts may be dried and stored for later use (after rehydration), orthey may be stored wet (e.g., in a buffer solution) for later orimmediate use. Cutting/fine dissection after de-masking 805′ may allowsignificantly higher yields while maintaining the highly specific natureand configuration of graft produced and allows cutting of only materialthat is deemed acceptable.

Any of the methods (including user interfaces) described herein may beimplemented as software, hardware or firmware, and may be described as anon-transitory computer-readable storage medium storing a set ofinstructions capable of being executed by a processor (e.g., computer,tablet, smartphone, etc.), that when executed by the processor causesthe processor to control perform any of the steps, including but notlimited to: displaying, communicating with the user, analyzing,modifying parameters (including timing, frequency, intensity, etc.),determining, alerting, or the like.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.For example, as used herein, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. It will be further understood that the terms“comprises” and/or “comprising,” when used in this specification,specify the presence of stated features, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, steps, operations, elements, components, and/orgroups thereof. As used herein, the term “and/or” includes any and allcombinations of one or more of the associated listed items and may beabbreviated as “/”.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if a device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of over and under. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and examples such as“comprises” and “comprising” means various components can be co-jointlyemployed in the methods and articles (e.g., compositions and apparatusesincluding device and methods). For example, the term “comprising” willbe understood to imply the inclusion of any stated elements or steps butnot the exclusion of any other elements or steps.

In general, any of the apparatuses and methods described herein shouldbe understood to be inclusive, but all or a sub-set of the componentsand/or steps may alternatively be exclusive, and may be expressed as“consisting of” or alternatively “consisting essentially of” the variouscomponents, steps, sub-components or sub-steps.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “about” or “approximately,” even if theterm does not expressly appear. The phrase “about” or “approximately”may be used when describing magnitude and/or position to indicate thatthe value and/or position described is within a reasonable expectedrange of values and/or positions. For example, a numeric value may havea value that is +/−0.1% of the stated value (or range of values), +/−1%of the stated value (or range of values), +/−2% of the stated value (orrange of values), +/−5% of the stated value (or range of values), +/−10%of the stated value (or range of values), etc. Any numerical valuesgiven herein should also be understood to include about or approximatelythat value, unless the context indicates otherwise. For example, if thevalue “10” is disclosed, then “about 10” is also disclosed. Anynumerical range recited herein is intended to include all sub-rangessubsumed therein. It is also understood that when a value is disclosedthat “less than or equal to” the value, “greater than or equal to thevalue” and possible ranges between values are also disclosed, asappropriately understood by the skilled artisan. For example, if thevalue “X” is disclosed the “less than or equal to X” as well as “greaterthan or equal to X” (e.g., where X is a numerical value) is alsodisclosed. It is also understood that the throughout the application,data is provided in a number of different formats, and that this data,represents endpoints and starting points, and ranges for any combinationof the data points. For example, if a particular data point “10” and aparticular data point “15” are disclosed, it is understood that greaterthan, greater than or equal to, less than, less than or equal to, andequal to 10 and 15 are considered disclosed as well as between 10 and15. It is also understood that each unit between two particular unitsare also disclosed. For example, if 10 and 15 are disclosed, then 11,12, 13, and 14 are also disclosed.

Although various illustrative embodiments are described above, any of anumber of changes may be made to various embodiments without departingfrom the scope of the invention as described by the claims. For example,the order in which various described method steps are performed mayoften be changed in alternative embodiments, and in other alternativeembodiments one or more method steps may be skipped altogether. Optionalfeatures of various device and system embodiments may be included insome embodiments and not in others. Therefore, the foregoing descriptionis provided primarily for exemplary purposes and should not beinterpreted to limit the scope of the invention as it is set forth inthe claims.

The examples and illustrations included herein show, by way ofillustration and not of limitation, specific embodiments in which thesubject matter may be practiced. As mentioned, other embodiments may beutilized and derived there from, such that structural and logicalsubstitutions and changes may be made without departing from the scopeof this disclosure. Such embodiments of the inventive subject matter maybe referred to herein individually or collectively by the term“invention” merely for convenience and without intending to voluntarilylimit the scope of this application to any single invention or inventiveconcept, if more than one is, in fact, disclosed. Thus, althoughspecific embodiments have been illustrated and described herein, anyarrangement calculated to achieve the same purpose may be substitutedfor the specific embodiments shown. This disclosure is intended to coverany and all adaptations or variations of various embodiments.Combinations of the above embodiments, and other embodiments notspecifically described herein, will be apparent to those of skill in theart upon reviewing the above description.

1-30. (canceled)
 31. A method of forming a compressible and compliantgraft from a porous bone, the method comprising: cutting donor bonetissue into a thin layer having a thickness of less than 5.5 mm; washingthe cut donor bone tissue with protease to remove organic material;defatting the cut donor bone tissue; applying a masking agent to a firstside of a body of the donor bone tissue to a thickness of less than 1.5mm, forming a masked portion and an unasked portion, wherein the maskingagent comprises a cyanoacrylate having a polar side chain including anoxygen; demineralizing the unmasked portion of the donor bone tissue;and removing the masking agent to form a graft comprising a first layerof demineralized bone extending a length and a width of the body; and asecond layer of mineralized bone that is continuously adjacent to thefirst layer, wherein the second layer has a thickness that is less than1.5 mm.
 32. The method of claim 31, wherein the masking agent comprisesethoxyethyl cyanoacrylate.
 33. The method of claim 31, wherein thecyanoacrylate having a polar side chain including an oxygen is selectedfrom the group consisting of ethoxyethyl cyanoacrylates, methoxypropylcyanoacrylates and ethoxymethyl cyanoacrylates.
 34. The method of claim31, wherein the cyanoacrylate having a polar side chain including anoxygen is selected from the group of cyanoacrylates having a lowblooming and relatively quick curing properties.
 35. The method of claim31, wherein the masking agent comprises a thickener to reduce viscosity.36. The method of claim 35, wherein the thickener comprises polymethylmethacrylate (PMMA).
 37. The method of claim 36, wherein a concentrationof PMMA in the masking agent is between about 4% and about 12%.
 38. Themethod of claim 36, wherein the PMMA comprises an average molecularweight of about 350,000 or less.
 39. The method of claim 31, whereindefatting the cut donor bone tissue comprises washing in acetone. 40.The method of claim 31, further comprising trimming the cut donor bonetissue to a length of between about 15 and 50 mm, and a width of betweenabout 10-25 mm.
 41. The method of claim 31, wherein applying the maskingagent comprises dipping the cut donor bone tissue into the masking agentfor less than 1 minute to a depth of less than 1.5 mm.
 42. The method ofclaim 31, further comprising drying the masking agent for 1 hour orless.
 43. The method of claim 31, wherein removing the masking agentcomprises rinsing the cut donor bone tissue in acetone.
 44. The methodof claim 31, further comprising dehydrating the graft for storage. 45.The method of claim 31, wherein the donor bone tissue comprisescancellous human bone.
 46. The method of claim 31, further comprisingconfirming a density or porosity of the cut donor bone tissue beforeapplying the masking agent.
 47. A method of forming a compressible andcompliant graft from a porous bone, the method comprising: cutting donorbone tissue into a thin layer having a thickness of less than 5.5 mm;removing organic material from the cut bone tissue; defatting the cutdonor bone tissue; applying a masking agent to a first side of a body ofthe donor bone tissue to a thickness of less than 1.5 mm, forming amasked portion and an unasked portion, wherein the masking agentcomprises a cyanoacrylate having a polar side chain including an oxygen,wherein the masking agent comprises PMMA at a concentration of betweenabout 4% and about 12%; demineralizing the unmasked portion of the donorbone tissue; and removing the masking agent to form a graft comprising afirst layer of demineralized bone extending a length and a width of thebody; and a second layer of mineralized bone that is continuouslyadjacent to the first layer, wherein the second layer has a thicknessthat is less than 1.5 mm.
 48. The method of claim 47, wherein themasking agent comprises ethoxyethyl cyanoacrylate.
 49. The method ofclaim 47, wherein the cyanoacrylate having a polar side chain includingan oxygen is selected from the group consisting of ethoxyethylcyanoacrylates, methoxypropyl cyanoacrylates and ethoxymethylcyanoacrylates.
 50. A method of forming a compressible and compliantgraft, the method comprising: cutting donor bone tissue into a thinlayer having a thickness of less than 5.5 mm; removing organic materialfrom the cut donor bone tissue; applying a masking agent to a first sideof the donor bone tissue to a thickness of less than 1.5 mm, forming amasked portion and an unmasked portion, wherein the masking agentcomprises an ethoxyethyl cyanoacrylate, wherein the masking agentcomprises PMMA having an average molecular weight of about 350,000 orless at a concentration of between about 4% and about 12%;demineralizing the unmasked portion of the donor bone tissue; andremoving the masking agent to form a graft comprising a body having alength, a width and a thickness that is less than half the length andhalf the width, the body further comprising: a first layer ofdemineralized bone extending the length and the width of the body; and asecond layer of mineralized bone that is continuously adjacent to thefirst layer, wherein the second layer has a thickness that is less than1.5 mm.